This invention relates to calibration of detection systems for non-elastically scattered light. In particular, the invention relates to the calibration of scanners for detection of biomolecules on microarrays.
Microarrays of biomolecules, such as DNA, RNA, cDNA, polynucleotides, oligonucleotides, proteins, and the like, are state-of-the-art biological tools used in the investigation and evaluation of biological processes, including gene expression and mutation for analytical, diagnostic, and therapeutic purposes. Microarrays typically comprise a plurality of polymers, e.g., oligomers, synthesized or deposited on a substrate in an array pattern of features. The support-bound polymers are typically called xe2x80x9cprobesxe2x80x9d, which function to bind or hybridize with a sample of polymer material under test, i.e., a moiety in a mobile phase (typically fluid), called a xe2x80x9ctargetxe2x80x9d in hybridization experiments. However, some investigators also use the reverse definitions, referring to the surface-bound polymers as targets and the solution sample of polymer as probes. Further, some investigators bind the target sample under test to the microarray substrate and put the polymer probes in solution for hybridization. Either of the xe2x80x9ctargetxe2x80x9d or xe2x80x9cprobesxe2x80x9d may be the one that is to be evaluated by the other (thus, either one could be an unknown mixture of polymers to be evaluated by binding with the other). All of these iterations are within the scope of this discussion herein. The plurality of probes and/or targets in each location in the array is known in the art as a xe2x80x9cfeaturexe2x80x9d. A feature is defined as a locus onto which a large number of probes and/or targets all having the same monomer sequence are immobilized. In use, the array surface is contacted with one or more targets under conditions that promote specific, high-affinity binding of the target to one or more of the probes. The targets are typically labeled with an optically detectable label, such as a fluorescent tag, dye or fluorophore, so that the targets are detectable with scanning equipment after a hybridization assay. DNA array technology, for example, offers the potential of using a multitude (hundreds of thousands) of different oligonucleotides to analyze changing mRNA populations.
Typical scanning equipment used for biomolecular analysis, such as scanning fluorometers, comprise an excitation light source, an optical system for directing light to and from a sample being scanned, a detection system and optionally an analysis system. To analyze a microchip after a hybridization assay, the scanner scans a light from its excitation light source across the microchip. The light excites the optically detectable labels on the hybridized biomolecules. The excited labels in turn emit light at one or more particular wavelengths. The emitted light from the biomolecules is detected and measured by the detection system and the measurements are analyzed by analysis equipment to determine the results of the assay. In competitive hybridization assays, more than one label may be used, each of which emit light having a characteristic emission spectrum, which may be narrow or broad, to distinguish the biomolecules on the microchip. The light emitted by each different label must be separately detected by the scanning equipment for analysis. State-of-the-art scanners are equipped with a detection system having multiple channels for detecting emissions at different wavelengths, for example. The detection systems having multiple channels are designed to detect signals from a combination of dyes or dyes having broad emission spectra that are used in labeling. Parameters such as the intensity, the wavelength, and the location of the emitted light on the microchip provide important information about the target material being assayed. Therefore, accurate measurement of these parameters is essential to providing accurate information about the target material.
The detection systems used in scanning equipment comprise one or more. detector components, such as photomultiplier tubes (PMTs). PMTs are known to age and to also deteriorate as a function of signals and overloads previously received.
An approach to improving the accuracy of a fluorometer used for scanning flow cells is to determine the relative fluorescence intensity or index (RFI) for a bulk sample in the flow cell. Gifford et al., U.S. Pat. Nos. 4,750,837 and 4,802,768, discuss and illustrate approaches for compensating for variations in excitation light using reference signals or reference paths, and the advantages and disadvantages of these approaches, and further, disclose a reference system that accounts for variations in signal levels from light sources and detection components that affect the RFI. A computation is made on measurements taken on the detection components and light sources, which indicates the relative concentration of the target being assayed using the flow cell. The computation is intended to cancel out the variations in the light sources and the detectors. The system described by Gifford et al. provides only a relative measurement of the target concentration in a bulk sample using flow cells. Therefore, there is little or no consistency between fluorometer systems taught by Gifford et al.
Thus, it would be advantageous to have a scanning system for scanning microarrays of biomolecules on microchips that is self-calibrating in that the sensitivity changes in the detection components are compensated for. Further, it would be advantageous to have a scanning system that could provide absolute target concentration measurement results where the results are reproducible from scanner to scanner. Still further, it would be advantageous if such self-calibration could be integrated into the scanner and the calibration be performed automatically.
The present invention provides a novel self-calibrating scanning system and method of calibrating which are useful for scanning microarrays of biomolecules on microchips. Since the present system is a scanning system that scans microarrays of minute quantities of biomolecules, a laser or other stable collimated light source is typically used as the excitation light source. The laser is characteristically very stable, such that the need to account for variations in excitation light on the system is essentially eliminated. The self-calibrating scanner and method compensate, and may also monitor, for changes in the component whose sensitivity will most likely to drift or vary with time and use, i.e., the detection components. Further, for scanning systems with multichannel detection components, the calibration portion of the invention can be made fairly redundant without much extra effort, if any. The self-calibrating scanner is particularly useful in scanning fluorometry of arrays. The self-calibrating scanner provides calibration capability of the specific sensitivities/scale factors in the detection color channels.
In one aspect of the invention, the self-calibrating scanning system comprises an excitation light source that produces an excitation light, an optics portion, a detection portion, and a calibration portion. The optics portion directs the excitation light from the excitation light source to a microarray of labeled biomolecules on a microchip that is under test. The detection portion comprises a detector that detects or measures emissions from labels on the microarray that are excited by the excitation light and produces an output signal responsive to the detected emissions. The calibration portion comprises a calibration apparatus and a compensation portion. The calibration apparatus comprises a calibration light source and optionally a calibration detector for ensuring a constant or calibrated light level from the calibration light source via a closed feedback control loop. The compensation portion comprises components to perform one or more of data collection, data storage, data comparison, data communication, and adjustment to the scanning system to compensate for any changes.
The calibration apparatus provides a calibrated light to the detection portion of the scanner. The detection portion measures the calibrated light and provides a corresponding output. The compensation portion measures or collects the output from the detection portion corresponding to the calibrated light and may compare the output to a stored reference value. If the output is different from the stored reference value, i.e., there is a change in detection sensitivity, the compensation portion compensates for the sensitivity change, either by adjusting the detection portion or providing sensitivity data for analysis. Such provided sensitivity data may be stored in a memory (for example, in association with data read from the array in response to excitation light). Any sensitivity data herein may be an indication of any change in sensitivity, so that such differences can be used in the processing of read data from an array to substantially compensate for the sensitivity changes, for example, which occurred between scanning different arrays. Alternatively, the sensitivity data may simply be an indication that sensitivity did not change, for example that between the scanning of arrays sensitivity remained constant, allowing a user to confidently compare results from arrays scanned at different times (or from different machines if any or no change in sensitivity with regard to a reference value is provided, or an absolute sensitivity value provided). The calibration portion can provide periodic calibration checks of the detection portion. The calibration portion may be a discrete unit or integral to the scanning system. The scanning system optionally further comprises an analysis portion. Alternatively, the scanning system may be otherwise associated with analysis equipment. The analysis portion comprises one or more of data collection, storage and analysis equipment components. The analysis portion receives the output signals from the detection portion and provides array data, such as the concentration of target in each location on the microarray. The analysis portion may further receive the sensitivity change data from the compensation portion, so that the sensitivity change data can be correlated with the array data.
The self-calibrating scanner of the invention compensates for sensitivity changes in the detection portion of the system to provide an absolute amount or concentration of target material on the microchip, not a relative amount, as in Gifford et al. (cited supra). Advantageously, the same microchip can be analyzed on other self-calibrating scanners of the present invention for the target concentration on the same microchip and the results will be substantially the same. In contrast, the system described by Gifford et al. essentially would not work for scanning minute quantities on arrays, and further, provides only relative target concentration values for bulk samples in a flow cell. Therefore, it is unlikely that any two or more systems described Gifford et al. will provide the same result for the same bulk sample/flow cell. The present invention does not determine target concentration on a relative scale. Advantageously, the present invention provides absolute results that are reproducible from one self-calibrating scanning system to another self-calibrating scanning system of the invention.
In another aspect of the invention, a method of calibrating a scanning system is provided. The method comprises the step of initially calibrating the detection portion of the scanner. The step of initially calibrating comprises the steps of generating a fixed signal (corresponding to the calibrated light level mentioned above) and measuring an output signal from the detection portion of the scanner in response to the fixed signal. The step of measuring may be repeated one or more times. The measurements are recorded and the mean value is calculated and stored as one reference value. The calibration apparatus and compensation portion described above may be used in the step of initially calibrating.
An additional aspect of the present invention may include simply retrieving from a memory stored sensitivity data and read data from an array, and correcting the read data based on the stored sensitivity data. Alternatively, another aspect may involve retrieving stored sensitivity data for respective different arrays (or different readings of the same array) and retrieving respective sensitivity data and, when the sensitivity data indicate no change in sensitivity, then comparing results from the different arrays (or different readings) or, when the sensitivity data indicates differences in sensitivity when the respective array readings were taken, then first correcting the read data from the different arrays (or different readings of the same array) then comparing the read results from them.
The method further comprises the step of subsequently calibrating the detection portion of the scanning system. The step of subsequently calibrating comprises the steps of separately generating another fixed signal that corresponds to the fixed signal mentioned above, using the calibration portion, as described above, measuring the corresponding output signal from the detection portion and comparing the corresponding output signal to the reference value, and compensating for any changes in sensitivity of the detection portion with respect to array data collected during a scan. The step of compensating comprises one or both of adjusting the detection portion of the scanning system to achieve a corresponding detection portion output signal with minimum deviation from the stored reference value, or providing detection sensitivity change data for analysis, so as to substantially compensate for any sensitivity changes for a scanned array. The step of subsequently calibrating may be repeated periodically and may be automatically or manually initiated. Moreover, implementation of the method of calibrating a scanning system in accordance with the invention can be automatic or manual. The present method helps to maintain throughput while reducing variations in detection sensitivity over time, such that the scanning system provides a substantially consistent accuracy level.